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Title Identification and sensory evaluation of flavour enhancers in Japanese traditional dried herring (Clupea pallasii) fillet

Author(s) Shah, A.K.M. Azad; Ogasawara, Masashi; Egi, Makoto; Kurihara, Hideyuki; Takahashi, Koretaro

Citation Food Chemistry, 122(1), 249-253https://doi.org/10.1016/j.foodchem.2010.02.072

Issue Date 2010-09

Doc URL http://hdl.handle.net/2115/44663

Type article (author version)

File Information article.pdf

Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

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Identification and sensory evaluation of flavour enhancers in Japanese traditional dried

herring (Clupea pallasii) fillet

A.K.M. Azad Shah a, *

, Masashi Ogasawara b

, Makoto Egi b

, Hideyuki Kurihara a, Koretaro

Takahashi a

a Division of Marine Life Science, Graduate School of Fisheries Sciences, Hokkaido

University, 3-1-1 Minato-cho, Hakodate, Hokkaido 041-8611, Japan

b Food Research & Development Laboratory, Kirin Kyowa Food Co. Ltd., 4041 Ami,

Ami-machi, Inashiki-gun, Ibaraki 300-0398, Japan

Running title: Flavour enhancers in dried herring fillet

*Corresponding author: Tel.: +81-138-40-5562; Fax: +81-138-40-5560

E-mail: azad_shh@yahoo.com (A.K.M. Azad Shah)

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Abstract

Flavour-enhancing components of dried herring fillet (migaki-nishin in Japanese) were

isolated and evaluated for their effects on sensory perception. Sensory evaluation revealed

that addition of dried herring fillet water-soluble extracts to Japanese noodle soup

significantly (P < 0.05) enhanced the soup flavour characters such as thickness, mouthfulness

and continuity. The extracts were fractionated by dialysis and chromatography. Fractions

containing flavour enhancers were isolated by sensory perception. Results from instrumental

analyses showed that the kokumi flavour enhancers in dried herring fillet were creatine and

creatinine.

Keywords: Herring; taste; kokumi; creatine; creatinine; sensory evaluation

1. Introduction

Dried herring (Clupea pallasii) fillet (DHF, Japanese, migaki-nishin) is a traditionally

popular food item in Japan due to its remarkable flavour-enhancing properties. In particular,

addition of DHF to noodle soup enhances flavour characters, such as thickness, mouthfulness

and continuity. These flavour characters are often called kokumi in Japanese. Compounds

added to enhance existing flavour characters, such as thickness, mouthfulness and continuity

of a food, without imparting a typical taste and aroma of their own, have been described as

kokumi flavour enhancers (Ueda, Sakaguchi, Hirayama, Miyajima, & Kimizuka, 1990; Ueda,

Yonemitsu, Tsubuku, Sakaguchi, & Miyajima, 1997).

The flavour of fish and shellfish principally originates from extractive components

(Konosu & Yamaguchi, 1982). In general, most flavour components of foods are

water-soluble. These include nucleotides, amino acids, peptides, organic acids and bases, and

inorganic ions. Other than amino acids, peptides and nucleotides, creatine, creatinine and

lactate were found to be taste-active components in dried skipjack (Fuke & Konosu, 1991).

Lactate, succinate, creatine, creatinine and hypoxanthine were also recognised as taste-active

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in stewed beef juice (Schlichtherle-Cerny & Grosch, 1998).

It is well known that amino acids and peptides contribute to the taste of a wide variety of

foods. The taste quality produced by amino acids and peptides has also been described as

kokumi that has translated as “rich thick taste” (Kawajiri, 1999). It is reported that pyrazines

and some peptides, generated together in certain foods during the process of boiling or aging

for a long period, form kokumi (Ogasawara, 2003). Moreover, Shima, Yamada, Suzuki, and

Harada (1998) have identified to be N-(1-methyl-4-hydroxy-3-imidazolin-2,2-ylidene)

alanine in beef broth as a “brothy taste” modifier, whereas glutathione enhanced the flavour

characteristics of thickness, mouthfulness and continuity when added to an umami solution

(Ueda et al., 1997). Recently, it has been reported that the addition of γ-glutamyl peptides to

a savoury matrix, e.g. sodium chloride and monosodium glutamate solutions or chicken broth,

significantly decreased the detection thresholds and remarkably enhanced mouthfulness,

complexity, and long lastingness of the savoury taste (Dunkel, Köster, & Hofmann, 2007).

Preliminary experiments showed that addition of DHF water-soluble extracts to Japanese

noodle soup (JNS) enhanced the soup flavour characters, such as thickness, mouthfulness

and continuity, and enhancement of these flavour characters increased with drying time

(Shah, Tokunaga, Kurihara, & Takahashi, 2008). However, to the best of our knowledge,

compounds responsible for the characteristic flavour-enhancing effects of DHF have not

been identified. Therefore, this study was aimed to identify the flavour-enhancing

compounds of DHF and to evaluate their effects on sensory perception.

2. Materials and methods

2.1. General

Field desorption mass spectra (FD-MS) were determined on a JEOL JMS-SX102A mass

spectrometer (JEOL Ltd., Japan). 1H and

13C nuclear magnetic resonance (NMR) spectra

were recorded on a Bruker AMX-500 NMR spectrometer (Bruker, Rheinstetten, Germany) at

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500 MHz and 126 MHz, respectively. Deuterium oxide (D2O) was used as solvent and

3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP-d4) was used as an internal standard.

2.2. Materials and chemicals

DHF was obtained from a fishery processing company, in Hakodate, Japan. Herring (C.

pallasii) was captured at the coast of Kamchatka Peninsula, Russia, in October 2006, and

kept frozen until it was processed. Upon arrival at the factory, herring was thawed, gutted,

washed and then filleted for drying. Herring fillets were dried using huge electric fans. Room

temperature and relative humidity were maintained at approximately 14 °C and 45%,

respectively. After drying, DHF was randomly sampled for analysis. The DHF was

comprised of 54.4% crude protein, 39.1% total lipid, and 4.3% ash on a dry weight basis. All

chemicals used were of analytical or HPLC grade and were purchased from Wako Pure

Chemical Industries Ltd. (Osaka, Japan), Sigma (St. Louis, MO), and Amersham Pharmacia

Biotech (Uppsala, Sweden).

2.3. Preparation of water-soluble extracts (WSE)

DHF was cut into small pieces and freeze-dried. The freeze-dried DHF (20 g) was then

defatted using n-hexane and homogenised with a 10-fold volume of de-ionised water. The

homogenate was centrifuged at 10,000 g for 20 min at 4 °C and supernatant was collected.

Resulting supernatant was extracted with ethanol (final concentration, 80%) to remove high

molecular weight compounds, followed by centrifugation and then filtration. After

evaporation and further freeze-drying of the filtrate, lyophilised powder was obtained as the

WSE of DHF (1.37 g, yield = 6.85%). In parallel, freeze-dried DHF (20 g) was boiled in a

10-fold volume of de-ionised water for 30 min and then homogenised. The homogenate was

centrifuged at 10,000 g for 20 min at 4 °C and supernatant was obtained. After ethanol

extraction and centrifugation, the obtained clear supernatant was freeze-dried to give the

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WSE of boiled DHF (1.78 g, yield = 8.90%).

2.4. Fractionation of the WSE of DHF

The WSE of DHF was dissolved in de-ionised water, and then the solution was dialysed

against de-ionised water using 1000 and 5000 Da cut-off membranes (Spectrum Laboratories,

Inc., California, USA) and finally freeze-dried. Three fractions were obtained, namely

fraction I (<MW 1000 Da), fraction II (MW 1000–5000 Da), and fraction III (>MW 5000

Da).

2.5. Gel filtration chromatography (GFC)

The fraction II (MW 1000–5000 Da) was dissolved into de-ionised water (100 mg/ml)

and, then applied onto the top of a water-cooled glass column (2.2 85.0 cm, Amicon

Corporation, Lexington, MA, England) filled with slurry of Sephadex G-25 (Amersham

Pharmacia Biotech, Uppsala, Sweden). The elution was achieved at a flow rate of 0.3 ml/min

at 4 °C with de-ionised water as the mobile phase to allow sensory evaluation of the

recovered fractions. The eluting solution was collected in 4 ml fractions. The UV absorbance

of each fraction was measured at 220 nm using a Hitachi U-2000 spectrophotometer (Hitachi

Ltd., Tokyo, Japan). The fractions were combined into five fractions F1–F5 on the basis of

elution profile. The individual fractions were freeze-dried and stored at –50 °C prior to use.

2.6. Separation by high-performance liquid chromatography (HPLC)

Fraction F3, thus obtained, was analysed on a Hitachi 655A HPLC system (Hitachi Ltd.,

Tokyo, Japan). Chromatographic separation was performed with a TSK-GEL G2000SW

semi-preparative column (10 µm; 7.5 × 300 mm, Tosoh Corp., Tokyo, Japan). The mobile

phase consisted of an aqueous solution of 80% acetonitrile containing 0.1% trifluoroacetic

acid. Elution was conducted at a flow rate of 1.0 ml/min and at room temperature. The

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eluting solution was monitored with UV absorbance at 214 nm. The obtained fractions were

then freeze-dried and stored at –50 °C until used for sensory evaluation and chemical

analysis.

2.7. Identification of the flavour enhancers in fraction F3-2

A fraction thus obtained by rechromatography, F3-2, was further rechromatographed by a

RP-HPLC system using an LC-10ATVP Shimadzu liquid chromatograph (Shimadzu Corp.,

Japan). Chromatographic separations were performed with an ODS column (Mightysil RP-18,

Kanto Chem. Co., Tokyo, Japan) either in analytical (5 µm; 4.6 × 250 mm, flow rate 0.4

ml/min) or semi-preparative scale (5 µm; 10 × 250 mm, flow rate 1.0 ml/min). A linear

gradient of acetonitrile from 10% to 20% for 20 min in water was used as mobile phase.

Fractions F3-2-A and F3-2-B were isolated as flavour enhancers. Subsequent MS and NMR

experiments of the isolates of RP-HPLC fractions F3-2-A and F3-2-B led to the identification

of the kokumi components as creatine and creatinine, respectively.

F3-2-A (Creatine): HRFDMS, m/z 132.0785 (MH+, calcd for C4H10N3O2, 132.0774);

1H

NMR (500 MHz, D2O), δ 3.98 (2H, s), 3.05 (3H, s); 13

C NMR (126 MHz, D2O), δ 177.0,

160.0, 56.4, 39.8.

F3-2-B (Creatinine): HRFDMS, m/z 114.0673 (MH+, calcd for C4H8N3O, 114.0668);

1H

NMR (500 MHz, D2O), δ 4.06 (2H, s), 3.05 (3H, s); 13

C NMR (126 MHz, D2O), δ 191.9,

172.2, 59.2, 33.0.

2.8. Sensory evaluation

Sensory evaluation was carried out by adding WSE and all other fractions (obtained by

various fractionation steps) to JNS, following the method of Ueda et al. (1997) with slight

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modification. JNS was prepared according to Shah, Tokunaga, Kurihara, and Takahashi

(2009). It was diluted with six volumes of distilled water and then subjected to sensory

evaluation. The WSEs of DHF and boiled DHF, and the three dialysed fractions obtained

from the WSE of DHF were dissolved in JNS at a concentration of 0.10%. The lyophilised

GFC and HPLC fractions were dissolved in JNS at concentration of 0.05% and 0.01%,

respectively. After addition of test samples to JNS, the solution was heated to 60 °C in a

water bath. About 50 ml of test and control solutions were served in opaque disposable

plastic cups at the same time. Panel members were instructed to put an adequate volume in

the mouth, and then to expectorate. The panelists were asked to judge the intensities of the

test samples using a scale of 17, where three points were assigned to the control solution.

Scoring was done on the basis of saltiness, umami, thickness, mouthfulness and continuity.

Sensory evaluation was performed in the separated sensory booths. The panel was composed

of three to five trained assessors (ages between 26 and 37 years) from the Food Research and

Development Laboratory, Kirin Kyowa Food Co. Ltd., Ibaraki, Japan. All the panelists were

trained in sensory experiments with various concentrations of commercial flavour enhancers

at regular intervals for at least 2 years and were, therefore, familiar with the techniques and

rating scales applied. Moreover, the members of the sensory panel had extensive experience

in tasting and agreed on the intensities of saltiness, umami, thickness, mouthfulness and

continuity in JNS.

2.9. Statistical analysis

Statistical analysis was performed using Microsoft Office Excel 2003. The student’s t-test

was carried out to determine significant differences between test samples and the control.

Trends were considered significant when the means of compared sets differed at P < 0.05.

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3. Results and discussion

3.1. Sensory characteristics of water-soluble extracts

The WSE had no basic taste but acquired a faint aroma in distilled water at a

concentration of 0.10% (data not shown). However, addition of the WSE of DHF to JNS

resulted in a pronounced effect of enhancing the soup flavour intensities of thickness,

mouthfulness and continuity, compared to the WSE of boiled DHF, whereas no significant (P

> 0.05) effect was observed on the saltiness and umami (Fig. 1). Sensory evaluation results

suggest that kokumi flavour enhancers are naturally produced in DHF. Therefore, the WSE of

DHF was used for further sensory-guided fractionation to identify the flavour enhancer.

The WSE of DHF was fractionated into three fractions by dialysis. Addition of these

three fractions to JNS showed no significant (P > 0.05) influence on basic taste such as

saltiness and umami (Table 1). However, addition of fraction II to JNS significantly (P <

0.05) increased the intensities of thickness and continuity of the soup flavour compared to the

control. Addition of fraction III significantly (P < 0.05) enhanced mouthfulness intensity,

while fraction I did not influence the soup flavour. From these results, it appears that fraction

II (MW 1000–5000 Da) might contain at least some of the flavour enhancers. It has been

reported that a peptide fraction (MW 1000–10,000) obtained from beef soup stock increased

mildness and mouthfulness when added to beef soup (Ishii et al., 1995). Ogasawara, Yamada,

and Egi (2006) also reported that peptide (MW 1000–5000 Da) products of Maillard

reactions contain key substances that give characteristic flavour (mouthfulness and

continuity) of miso ripened for extended periods.

3.2. Chromatographic separation of fraction II

Fraction II (MW 1000–5000 Da) was further fractionated using gel filtration

chromatography. The eluting fractions were separated into five fractions F1–F5 (Fig. 2).

Fractions F4 and F5 were recovered in small amounts, therefore only three fractions F1–F3

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were subjected to further analysis. The flavour-enhancing effect was predominant in F3,

followed by F2, whereas F1 had practically no influence on the soup flavour (Table 2).

Fraction F3 showed significant (P < 0.05) enhancement of thickness and continuity of the

JNS compared to control.

Fraction F3 was divided into four fractions, from F3-1 to F3-4 on semi-preparative HPLC,

as shown in Fig. 3. The taste profiles of JNS containing the fractions from F3-1 to F3-4 are

shown in Table 3. Addition of those individual fractions to JNS had no significant influence

on basic taste qualities, such as saltiness and umami, compared to the control. However,

fraction F3-2 significantly (P < 0.05) enhances the intensities of mouthfulness and continuity

of the JNS compared to the control. Furthermore, sensory evaluation showed that, not only

mouthfulness and continuity, but also thickness were more clearly recognised by the panelists

in fraction F3-2 than in other fractions.

3.3. Isolation and identification of flavour enhancers in fraction F3-2

Fraction F3-2 was further fractionated by RP-HPLC, to afford compounds F3-2-A and

F3-2-B (Fig. 4). The MS and NMR data revealed that the structure of flavour-enhancing

compounds of F3-2-A and F3-2-B were creatine (Hanstock, Rothman, Prichard, Jue, &

Shulman, 1988) and creatinine (Yamada, Akahori, & Matsuda, 2009), respectively. The

identification of both compounds was further confirmed by comparison of spectroscopic and

chromatographic data with those obtained for the corresponding reference compound.

Although creatine and creatinine have already been identified as taste-active compounds in

dried skipjack (Fuke & Konosu, 1991), Vietnamese fish sauce (Park, Watanabe, Endoh,

Watanabe, & Abe, 2002), and stewed beef juice (Schlicherle-Cerny & Grosch, 1998), the

flavour-enhancing activity of these compounds has not so far been reported.

Although molecular weight of creatine and creatinine are 131.13 and 113.11 Da,

respectively, surprisingly, these two compounds were found in fraction II (MW 1000–5000

10

Da). To determine the possible reasons for this, we dialysed a solution of authentic creatine

and creatinine using a MW 1000 Da cut-off membrane. After dialysing these compounds

against de-ionised water, it was found that the dialysis tube retained (i.e. >MW 1000 Da)

58% and 8% of creatine and creatinine, respectively. Moreover, creatine has positive and

negative ions; aggregation of molecules in solution can therefore be expected. Thus creatine

and creatinine were isolated from fraction II (MW 1000–5000 Da).

3.4. Sensory evaluation of creatine and creatinine

To confirm the roles of creatine and creatinine as kokumi flavour enhancers, food grade

creatine and creatinine were added to the JNS and presented to the trained sensory panelists.

Weight percentages of creatine and creatinine, of fraction F3-2-A and F3-2-B, were

calculated from representative peak areas and the ratio of those were approximated as 95%

and 5%, respectively. Test samples were added to the JNS at a concentration of 0.01%.

Sensory evaluation results revealed that the addition of creatine and creatinine to the JNS

significantly (P < 0.05) increased the intensities of thickness, mouthfulness and continuity of

the JNS compared to the control, while no significant effect was observed on basic taste

qualities, such as saltiness and umami (Fig. 5). Comparing the flavour profiles of creatine

and creatinine with that of the HPLC fraction F3-2 (Table 3) demonstrated that there was no

significant (P > 0.05) difference between the individual flavour characteristics, thus

indicating that creatine and creatinine might have made a contribution to kokumi

enhancement of DHF. It has been reported that addition of sulphur-containing components,

such as, trans-S-propenyl-L-cysteine sulfoxide and γ-glutamyl-trans-S-propenyl-L-cysteine

sulfoxide, to an umami solution gave a characteristic kokumi flavour (Ueda, Tsubuku, &

Miyajima, 1994). The multivalent taste-modulating Maillard reaction product, alapyridaine,

in beef broth, enhances sweetness and umami characters (Ottinger & Hofmann, 2003), and,

most recently, γ-L-glutamyl dipeptides have been identified as key kokumi molecules,

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enhancing mouthfulness and complex taste continuity of matured Gouda cheese (Toelstede,

Dunkel, & Hofmann, 2009).

4. Conclusion

Flavour-enhancing compounds, creatine and creatinine, were isolated and identified from

water-soluble extracts of dried herring fillet. Sensory evaluation showed that creatine and

creatinine enhanced the characteristic flavours of thickness, mouthfulness and continuity of

the JNS, thus indicating that creatine and creatinine might be the kokumi flavour enhancers of

dried herring fillet.

Acknowledgements

The authors thank Eri Fukushi and Kenji Watanabe (GC-MS and NMR Laboratory,

Faculty of Agriculture, Hokkaido University) for measurement of MS and NMR spectra. The

authors also thank the members of the sensory panel of the Food Research and Development

Laboratory, Kirin Kyowa Food Co. Ltd., Ibaraki, Japan, for their help with sensory

assessments.

References

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juice. Zeitschrift für Lebensmittel -Untersuchung und -Forschung A, 207, 369–376.

Shah, A. K. M. A., Tokunaga, C., Kurihara, H., & Takahashi, K. (2008). Changes in chemical

composition and sensory properties of migaki-nishin (dried herring fillet) during drying.

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their contribution to the taste of migaki-nishin (dried herring fillet) during drying. Food

Chemistry, 115, 1011–1018.

Shima, K., Yamada, N., Suzuki, E., & Harada, T. (1998). Novel brothy taste modifier isolated

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from beef broth. Journal of Agricultural and Food Chemistry, 46, 1465–1468.

Toelstede, S., Dunkel, A., & Hofmann, T. (2009). A series of kokumi peptides impart the

long-lasting mouthfulness of matured Gouda cheese. Journal of Agricultural and Food

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Figure Captions

Fig. 1. Taste profile of Japanese noodle soup (JNS) treated with water-soluble extracts (WSE)

of dried herring fillet (DHF).

(–) JNS with a score of three for control, (–o–) JNS treated with the WSE of DHF, (---) JNS

treated with the WSE of boiled DHF.

Fig. 2. Gel filtration elution profile of fraction II.

Fig. 3. HPLC chromatogram of fraction F3.

Fig. 4. RP-HPLC chromatogram of fraction F3-2.

Fig. 5. Taste performance of creatine and creatinine in Japanese noodle soup (JNS) by

sensory evaluation.

(---) JNS with a score of three for control; (−▲−) JNS treated with a combination of

creatine/creatinine (95/5).

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Table 1

Taste profile of Japanese noodle soup (JNS) containing water-soluble fractions obtained by

stepwise dialysis a

Attribute Fraction I Fraction II Fraction III

Salty 3.1 ± 0.54 3.0 ± 0.71 3.4 ± 1.14

Umami 3.3 ± 0.45 4.1 ± 0.89 4.0 ± 0.79

Thickness 3.7 ± 0.84 4.7 ± 0.67* 4.0 ± 0.79

Mouthfulness 3.7 ± 0.83 4.7 ± 0.97 4.7 ± 0.84*

Continuity 3.6 ± 0.89 4.9 ± 0.54* 4.6 ± 1.08

a Data are expressed as means ± standard deviation (n = 5). Taste intensity scored on a 1–7

point scale where three points were assigned to the score of JNS.

* Asterisks indicate significant difference from control at P < 0.05.

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Table 2

Taste profile of Japanese noodle soup (JNS) containing fractions obtained by gel filtration

chromatography a

Attribute F1 F2 F3

Salty 4.0 ± 0.71 4.25 ± 0.43 4.38 ± 0.41

Umami 3.75 ± 0.83 4.0 ± 0.71 4.38 ± 0.65

Thickness 3.5 ± 0.87 4.13 ± 0.54 4.5 ± 0.50*

Mouthfulness 3.5 ± 0.87 4.5 ± 0.5* 4.38 ± 0.65

Continuity 3.25 ± 0.43 3.63 ±0.65 4.75 ± 0.83*

a Data are expressed as means ± standard deviation (n = 4). Taste intensity scored on a 1–7

point scale where three points were assigned to the score of JNS.

* Asterisks indicate significant difference from control at P < 0.05.

17

Table 3

Taste profile of Japanese noodle soup (JNS) containing fractions from F3-1 to F3-4 a

Attribute F3-1 F3-2 F3-3 F3-4

Salty 3.0 ± 0 3.0 ± 0 3.0 ± 0 2.83 ± 0.29

Umami 3.0 ± 0 3.0 ± 0 3.17 ± 0.29 3.17 ± 0.29

Thickness 3.17 ± 0.29 4.33 ± 1.04 3.67 ± 0.76 3.33 ± 0.58

Mouthfulness 3.0 ± 0 3.83 ± 0.29* 3.5 ± 0.5 3.5 ± 0.87

Continuity 3.67 ± 0.76 4.17 ± 0.76* 3.67 ± 0.76 3.33 ± 0.58

a Data are expressed as means ± standard deviation (n = 3). Taste intensity scored on a 1–7

point scale where three points were assigned to the score of JNS.

* Asterisks indicate significant difference from control at P < 0.05.

Fig. 1 (Shah et al.)

0

2

4

salty

umami

thicknessmouthfulness

continuity

Fig. 2 (Shah et al.)

0.00.20.40.60.81.01.21.41.6

1 16 31 46 61 76 91 106 121

Abs

orba

nce

at 2

20 n

m

Fraction number (4 ml/fraction)

F1

F2

F3

F4

F5

Fig. 3 (Shah et al.)

5 10 15 20 25 30

F3-2

F3-4F3-1

F3-3

Abs

orba

nce

at 2

14 n

m

Retention time (min)

Fig. 4 (Shah et al.)

Abs

orba

nce

at 2

14 n

m F3-2-A

F3-2-B

Retention time (min)

Fig. 5 (Shah et al.)

0

2

4

salty

umami

thicknessmouthfulness

continuity